PROJECT SUMMARY: In this project, we have discovered using intravital microcopy imaging in a humanized mouse model of sickle cell disease (SCD) that activated neutrophils play a direct role in vaso-occlusive crisis (VOC) by interacting with circulating erythrocytes (sRBC). We have described heterogeneity in the ability of neutrophils to capture sRBC and recently ascribed it to their chronological aging in the circulation and the exposure to the microbiota. Indeed, microbiota depletion markedly reduces aged neutrophil counts, and improves the acute and chronic SCD complications. Preliminary data using a model of psychological stress, a known VOC trigger, suggest that stress- induced exacerbation of VOC also depends on the microbiota and aged neutrophil generation promoted by the IL-17A/G-CSF pathway. In this funding period, we propose to investigate the innovative hypothesis that the microbiota critically regulates SCD activity. In Specific Aim 1, we will evaluate how the microbiota modulates psychological stress-induced sickle cell VOC. We will assess the mechanisms of stress signals linking the brain to the immune response, focusing on neural (sympathetic nervous system) and stress hormones (glucocorticoids). We will identify the source of IL-17A elicited by stress, evaluate its function using IL-17A- deficient mice, and the mechanisms by which the microbiota activates the IL-17A/G-CSF pathway. In Specific Aim 2, we will define the role of neutrophils and microbiota in chronic sickle cell-induced end-organ damage. We have found that depletion of the microbiota markedly improved the chronic organ damage in SCD mice. Whether the microbiota mediates organ damage through interactions with leukocytes or other targets is unclear. We will investigate the role of neutrophils in organ damage in G-CSF-deficient mice which are neutropenic. We will investigate whether the microbiota signals to hematopoietic or non-hematopoietic cells using conditional Myd88- deletion. Since our preliminary data suggest that microbiota depletion significantly reduces iron deposition in tissues, we will investigate the role of iron chelation therapy in SCD mice. In collaboration with Dr. Craig Branch, we will monitor the impact of microbiota depletion by T2* magnetic resonance imaging and test the potential of siderophore probiotics. In Specific Aim 3, we will develop new strategies to harness the microbiota for SCD treatment. We will characterize the differential effect of antibiotics using 16S rDNA sequencing. We will manipulate the microbiota with probiotics and investigate specifically the role of segmented filamentous bacteria (SFB), as they are known to induce IL-17A/G-CSF, using fecal transplantations in vancomycin-treated or germ- free SCD mice. Since hydroxyurea (HU) has antimicrobial activity that can significantly alter the microbiome, we will evaluate its effect on gut microbiota by 16S rDNA sequencing and determine using fecal transplant the contribution of HU-modified microbiota in disease activity. These studies will provide insight on a new mechanism regulating SCD manifestations and lead to novel ways to target inflammation in this disease.